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Divergence of gut permeability and mucosal immune gene expression in twogluten-associated conditions: celiac disease and gluten sensitivity

BMC Medicine 2011, 9:23 doi:10.1186/1741-7015-9-23

Anna Sapone (annasapone@yahoo.it)Karen M Lammers (klammers@mbrc.umaryland.edu)

Vincenzo Casolaro (casolaro@jhmi.edu)Marcella Cammarota (marcella.cammarota@unina2.it)

Maria T Giuliano (mariateresa.giuliano@unina2.it)Mario De Rosa (mario.derosa@unina2.it)

Rosita Stefanile (rstefanile@isa.cnr.it)Giuseppe Mazzarella (gmazzarella@isa.cnr.it)

Carlo Tolone (carlo.tolone@unina2.it)Maria I Russo (mariaitria.russo@unina2.it)

Pasquale Esposito (pasquale.esposito@unina2.it)Franca Ferraraccio (franca.ferraraccio@unina2.it)

Maria Carteni (maria.carteni@unina2.it)Gabriele Riegler (gabriele.riegler@unina2.it)

Laura de Magistris (laura.demagistris@unina2.it)Alessio Fasano (afasano@mbrc.umaryland.edu)

ISSN 1741-7015

Article type Research article

Submission date 21 January 2011

Acceptance date 9 March 2011

Publication date 9 March 2011

Article URL http://www.biomedcentral.com/1741-7015/9/23

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1

Divergence of gut permeability and mucosal immune gene expression in two gluten-

associated conditions: celiac disease and gluten sensitivity

Anna Sapone1,2

, Karen M. Lammers2, Vincenzo Casolaro

2,3, Marcella Cammarota

4, Maria Teresa

Giuliano4, Mario De Rosa

4, Rosita Stefanile

5, Giuseppe Mazzarella

5, Carlo Tolone

6, Maria Itria

Russo7, Pasquale Esposito

7, Franca Ferraraccio

8, Maria Cartenì

4, Gabriele Riegler

1, Laura de

Magistris1, Alessio Fasano

2,*

1Department of Internal and Experimental Medicine Magrassi-Lanzara, Seconda Università degli

Studi di Napoli, Naples, Italy

2Mucosal Biology Research Center, University of Maryland School of Medicine, Baltimore,

MD, USA

3Johns Hopkins Asthma and Allergy Center, Johns Hopkins University School of Medicine,

Baltimore, MD, USA

4Department of Experimental Medicine, Seconda Università di Napoli, Naples, Italy

5Institute of Food, Consiglio Nazionale delle Ricerche (CNR), Avellino, Italy

6Department of Pediatrics, Seconda Università degli Studi di Napoli, Naples, Italy

7Servizio di Endoscopia Digestiva, Seconda Università degli Studi di Napoli, Naples, Italy

8Morfopatologia, Seconda Università degli Studi di Napoli, Naples, Italy

*Corresponding author

Alessio Fasano

Email: afasano@mbrc.umaryland.edu

2

Abstract

Background: Celiac disease (CD) is an autoimmune enteropathy triggered by the ingestion of

gluten. Gluten-sensitive individuals (GS) cannot tolerate gluten and may develop gastrointestinal

symptoms similar to those in CD, but the overall clinical picture is generally less severe and is

not accompanied by the concurrence of tissue transglutaminase autoantibodies or autoimmune

comorbidities. By studying and comparing mucosal expression of genes associated with

intestinal barrier function, as well as innate and adaptive immunity in CD compared with GS, we

sought to better understand the similarities and differences between these two gluten-associated

disorders.

Methods: CD, GS and healthy, gluten-tolerant individuals were enrolled in this study. Intestinal

permeability was evaluated using a lactulose and mannitol probe, and mucosal biopsy specimens

were collected to study the expression of genes involved in barrier function and immunity.

Results: Unlike CD, GS is not associated with increased intestinal permeability. In fact, this was

significantly reduced in GS compared with controls (P = 0.0308), paralleled by significantly

increased expression of claudin (CLDN) 4 (P = 0.0286). Relative to controls, adaptive immunity

markers interleukin (IL)-6 (P = 0.0124) and IL-21 (P = 0.0572) were expressed at higher levels

in CD but not in GS, while expression of the innate immunity marker Toll-like receptor (TLR) 2

was increased in GS but not in CD (P = 0.0295). Finally, expression of the T-regulatory cell

marker FOXP3 was significantly reduced in GS relative to controls (P = 0.0325) and CD patients

(P = 0.0293).

Conclusions: This study shows that the two gluten-associated disorders, CD and GS, are

different clinical entities, and it contributes to the characterization of GS as a condition

3

associated with prevalent gluten-induced activation of innate, rather than adaptive, immune

responses in the absence of detectable changes in mucosal barrier function.

4

Background

Gluten is the structural protein component of the grains wheat, rye and barley, which are the

basis for a variety of flour- and wheat-derived food products consumed throughout the world.

Possibly, the introduction of gluten-containing grains, which occurred about 10,000 years ago

with the advent of agriculture, represented a “mistake of evolution” that created the conditions

for human diseases related to gluten exposure, the best known of which are mediated by the

adaptive immune system: wheat allergy and celiac disease (CD). In both conditions, the reaction

to gluten is mediated by T-cell activation in the gastrointestinal mucosa. However, in wheat

allergy, it is the cross-linking of immunoglobulin E (IgE) by repeat sequences in gluten peptides

(for example, Ser-Gln-Gln-Gln-(Gln-)Pro-Pro-Phe) that triggers the release of chemical

mediators, such as histamine, from basophils and mast cells [1]. In contrast, CD, which affects

approximately 1% of the general population, is an autoimmune disorder, as heralded by the

appreciation of specific serologic markers, most notably serum antitissue transglutaminase (tTG)

autoantibodies, by the autoimmune enteropathy that characterizes this condition and by

autoimmune comorbidities.

Besides CD and wheat allergy, there are cases of gluten reactions in which neither

allergic nor autoimmune mechanisms are involved. These are generally defined as gluten

sensitivity (GS) [2-5]. Some individuals who experience distress when eating gluten-containing

products and show improvement when following a gluten-free diet may have GS instead of CD.

GS patients are unable to tolerate gluten and develop an adverse reaction when eating gluten that

usually, and differently from CD, does not lead to small intestinal damage. While the

gastrointestinal symptoms in GS may resemble those associated with CD, the overall clinical

picture is generally less severe and is not accompanied by the concurrence of tTG autoantibodies

5

or autoimmune disease. Typically, the diagnosis is made by exclusion, and an elimination diet

and “open challenge” (that is, the monitored reintroduction of gluten-containing foods) are most

often used to evaluate whether the patient’s health improves with the elimination or reduction of

gluten from the diet.

A number of in vitro studies have confirmed the cytotoxicity of gluten’s main antigen,

gliadin. Gliadin has agglutinating activity, reduces F-actin content, inhibits cell growth, induces

apoptosis, alters redox equilibrium and causes a rearrangement of the cytoskeleton through the

zonulin pathway and the loss of tight junction (TJ) competence in the gastrointestinal mucosa [6-

9]. The diversity of gluten-induced conditions is in line with the notion that the immune system

reacts to and deals with the triggering environmental factor, gliadin, in distinct ways. In the

present study, we sought to gain initial knowledge on intestinal barrier function and the immune

response to gluten in patients with GS. Specifically, we were interested in understanding to what

extent innate and adaptive immune pathways are activated in GS compared to CD. To achieve

these aims, we looked at the mucosal expression of genes associated with intestinal barrier

function and immune parameters known or implied to be aberrantly regulated in CD. The results

provide the first documentation to date of genes and pathways possibly involved in the

pathogenesis of GS, and, at the same time, contribute to improving our understanding of the

processes leading to CD and other autoimmune phenomena.

Methods

Definition of GS

GS patients are defined as those patients in which CD, wheat allergy and other clinically

overlapping diseases (type 1 diabetes, inflammatory bowel diseases and Helicobacter pylori

6

infection) have been ruled out and whose symptoms were triggered by gluten exposure and

alleviated by gluten withdrawal. All enrolled patients underwent a gluten challenge carried out

for approximately 4 months under clinical supervision. At the end of the challenge, patients

underwent CD serology screening, Human Leukocyte Antigen (HLA) genes DQ2/DQ8 typing

and an upper endoscopy with duodenal biopsies. Once endoscopies were performed, patients

were placed back on a gluten-free diet and their symptoms monitored over time. GS were

considered those patients with negative autoantibody serology (endomysium antibodies-

immunoglobulin A (EMA-IgA) and tTG-IgA), normal mucosa (Marsh stage 0) or increased

intraepithelial lymphocytes (Marsh stage 1) and improvement of symptoms within days of the

implementation of the diet. To avoid any possible selection bias and to prove that these patients

are different from CD patients, we elected to enroll every patient fulfilling the above-described

definition of gluten sensitivity.

Subjects

Consent was obtained from all enrolled subjects after the nature of the investigation was

explained and in accordance with the approved protocol from the Institutional Review Board at

the University of Naples. A total of 26 GS patients diagnosed according to the criteria outlined

above were enrolled. For comparison, 42 patients with active CD were recruited according to the

modified 2004 criteria of the European Society of Pediatric Gastroenterology, Hepatology and

Nutrition (ESPGHAN) [10]. Finally, 39 control subjects were enrolled from among individuals

undergoing upper endoscopy for dyspepsia. We limited our control group to individuals who had

exclusively dyspeptic symptoms and no underlying inflammation as proven by normal

erythrocyte sedimentation rate, C-reactive protein and mucoprotein evaluation. These subjects

7

did not have CD or GS and are referred to hereinafter as dyspeptic controls (DC).

Subject characteristics are summarized in Table 1. For each set of experiments, a representative

subgroup of CD, GS and DC were analyzed on the basis of the subjects’ consent to participate

in part or all of the proposed studies and on material transfer agreements between the

institutions involved in this project. At least six duodenal biopsies were collected for

histological evaluation and gene expression. No selection bias based on sex, age or type of

symptoms was observed.

Determination of intestinal permeability

In vivo permeability was determined by means of the lactulose/mannitol (LA/MA) test as

previously described [11]. The detection and measurement of the two sugar probes in the urine

was performed by high-performance anion exchange chromatography coupled with pulsed

amperometric detection, which permits direct quantification of nonderivative carbohydrates on a

Dionex model DX-500 with a gradient pump module GP40 and sample loop of 50 µl. Samples

were loaded onto CarboPac PA-100 guard columns and eluted with a NaOH-NaAc gradient

(Dionex, Sunnyvale, California, U.S.A.).

Histology and immunohistochemistry of jejunal biopsies

Serial sections (4 µm) were prepared from duodenal formalin-fixed, paraffin-embedded biopsies.

Biopsy specimens were staged by histology according to the Marsh classification [12, 13].

Immunostaining to identify intraepithelial lymphocytes (IELs) was performed as described

elsewhere [14]. Acetone-fixed sections (5 µm) were stained with monoclonal antibodies for CD3

8

(1:200; Dako, Milan, Italy) and T cell receptor γδ (TCRγδ) (1:50; Thermo Scientific) using the

peroxidase-antiperoxidase protocol. The sections were finally stained with Mayer’s hematoxylin

(Sigma- Aldrich) , St. Louis, Missouri, U.S.A. IEL density was calculated as the percentage of

enterocytes (ECs). All slides were analyzed by two observers who were blinded to these

procedures.

Determination of mucosal gene expression

Intestinal gene expression was measured by quantitative real-time polymerase chain reaction

(qPCR) assay as described previously [15]. Small intestinal biopsies from participating subjects

were homogenized and total RNA was extracted using TRIzol reagent (Invitrogen, Grand Island,

NY, USA). Primers and probes for qPCR of specific transcripts and of the 18S gene as a

housekeeping control were purchased from Applied Biosystems (Foster City, CA, USA). qPCR

was performed using the TaqMan protocol and the Applied Biosystems 7500 Fast Real-Time

PCR System. Amplification conditions were as follows: 50°C for 2 minutes and 95°C for 10

minutes, followed by 50 cycles at 95°C for 15 seconds and 60°C for 1 minute. The data were

calculated as the change in cycle threshold (∆CT) of the gene of interest to 18S and are expressed

as percentages of 18S (that is, 2-∆CT

× 100).

Statistical analysis

All data were analyzed and graphed using Prism version 5.0 software (GraphPad Software, La

Jolla, CA, USA). Most of the variables examined in this study appeared to be arranged in a non-

normal, positively skewed distribution, making conventional parametric statistics misleading.

Therefore, data are described by the medians and interquartile ranges (IQRs), and the degree of

9

variability among groups was determined using the Kruskal-Wallis nonparametric algorithm. A

two-tailed Mann-Whitney U test (MWU) was used for pairwise comparisons. The level of

significance was set at P < 0.05.

Results

Clinical and laboratory characteristics of GS subjects

The patients who fulfilled the GS diagnostic criteria (see Methods section) experienced

symptoms overlapping those presented by CD patients (see Table 1), but their symptoms

resolved within a few days after the implementation of the gluten-free diet, and they remained

symptom-free for the entire follow-up period (up to 4 years). Interestingly, 48% of GS patients

were anti-gliadin antibody (AGA)-positive, and 57% were HLA-DQ2-positive and/or HLA-

DQ8-positive. Fifty-six percent of AGA-positive GS patients were HLA-DQ2-positive and/or

HLA-DQ8-positive, while the remaining 44% were HLA-negative, suggesting that AGA

production was not associated with a HLA-DQ2-restricted and/or HLA-DQ8-restricted

presentation.

Intestinal permeability

While CD is consistently associated with impaired mucosal barrier function and increased small

intestinal permeability, it is not known whether patients with GS present similar alterations. To

address this question, the LA/MA urinary ratio was determined in the three study groups. The

LA/MA ratio, and hence small intestinal permeability, varied significantly among the three

groups (P = 0.0113; Kruskal-Wallis test). In particular, it was significantly higher in CD patients

compared to GS patients (P = 0.0138; MWU). A similar difference between CD patients and DC

10

only approximated significance (P = 0.0950). Interestingly, the LA/MA ratio in GS patients was

also significantly reduced relative to DC (P = 0.0308) (Figure 1).

Figure 1 shows that the increase in intestinal permeability in CD patients, relative to DC,

was ascribed to a significantly increased urinary concentration of lactulose (P = 0.0400), with no

significant changes in mannitol titers (P = 0.4937; not significant). Since the urinary recovery of

lactulose reflects its exclusive transport through the paracellular pathway, while mannitol is a

marker of the transcellular pathway [11], this confirms that the changes in LA/MA ratio

observed in CD reflect increased paracellular permeability. Likewise, the reduced LA/MA ratio

in GS patients relative to CD and DC appeared to be exclusively determined by reduced

lactulose titers, a finding again in line with the involvement of processes regulating

macromolecular passage through the paracellular pathway. Specifically, the median percentage

of urinary lactulose in GS was significantly lower than that in CD (P = 0.0049), while the

difference between GS and DC was not significant (P = 0.1817; not significant).

Small intestinal expression of TJ proteins

To understand whether the changes in intestinal permeability observed in CD and GS are

associated with altered expression of genes encoding for key TJ components, the expression of

claudins (CLDNs), TJ protein (TJP)-1 (also known as zonula occludens-1) and occludin (OCLN)

was determined in intestinal tissues obtained by upper gastrointestinal endoscopy. Biopsy

specimens were immediately processed for mRNA extraction, and the transcript levels of the

genes CLDN1, CLDN2, CLDN3, CLDN4, TJP1 and OCLN were measured by qPCR. As shown

in Figure 2, CLDN4 was expressed at significantly higher levels in GS than in CD (P = 0.0286),

while a similar difference with DC almost reached significance (P = 0.0565). Similar levels of

11

CLDN4 were detected in biopsies from DC and CD (P = 0.7279; not significant). Likewise, no

significant differences were observed across the three groups in the mRNA levels of CLDN1 or

CLDN2 or the other TJ-related genes examined (not shown). Although this might be due to the

limited number of cases studied, it is unlikely that inclusion of a larger sample would

compensate for the high level of variability and skewness encountered within each group.

Intraepithelial lymphocytes

The GS patients recruited in this study did not present relevant autoimmune phenomena and

autoimmune serology (Table 1). In addition, in this group of patients, we did not detect a clear-

cut association with the major histocompatibility complex (MHC) haplotype, as extensively

documented in CD patients. These clinical findings led us to hypothesize that the adaptive

immune system may not be as critically involved in GS as it is in CD (or in wheat allergy). To

substantiate this argument, our end points included markers of adaptive and innate immune

responses in the small intestinal mucosa of these subjects. In a first series of observations, we

compared the numbers of IELs in GS versus CD patients. As recently reported by our group [15],

histology revealed a normal to mildly inflamed mucosa (Marsh stage 0 or 1) in GS patients,

while all CD patients showed partial or subtotal villous atrophy with crypt hyperplasia according

to the ESPGHAN criteria [10]. The results of CD3 immunohistochemistry are summarized in

Figure 3. The numbers of CD3+ IELs varied considerably among the three groups (P < 0.0001;

Kruskal-Wallis test). As anticipated, CD patients had increased numbers of CD3+ IEL relative to

DC (P < 0.0001; MWU). CD3+ IELs were significantly more numerous in GS patients than in

DC (P < 0.0001), but significantly less so than in CD (P = 0.0012). Notably, though, all but four

GS patients had IEL numbers above the currently accepted range of normality (≤30/100 ECs),

12

suggesting an intermediate-level, yet pathogenically significant, involvement of the adaptive

immune system in this condition. On the other hand, the number of TCRγδ IELs was consistently

elevated in CD (>3.4/100 ECs), while the numbers in GS and DC were similar (data not shown).

Mucosal expression of adaptive immunity-related cytokines

A number of studies have documented the gluten-dependent rise in the systemic and mucosal

expression of cytokines associated with Th1 and Th17 adaptive responses in CD [16-19]. We

have recently shown that the Th17 signature cytokine, IL-17A, is expressed at significantly

higher levels in the small intestinal mucosa of CD patients but not GS patients [15]. We have also

shown that gliadin induces circulating monocytes to produce the Th17-active cytokine, IL-23, an

effect mediated by concomitant expression of IL-1β and IL-6 [17, 20]. To extend these

observations and to further define the role of adaptive immunity in GS, mRNA extracted from

small intestine biopsies was subjected to qPCR for expression of the pleiotropic, Th17-activating

cytokine interleukin (IL)-6, the Th1 cytokine interferon (IFN)-γ, and IL-21, a Th1 cytokine

involved both in the differentiation of Th17 cells and in sustaining ongoing Th1 responses [21].

As shown in Figure 4, CD patients as a group expressed higher levels of transcripts for IL-6

(IL6), IFN-γ (IFNG) and IL-21 (IL21) relative to DC. However, because of a high degree of

variability among CD donors and because of a subgroup effect already documented in our earlier

report on IL-17A [15], only the difference in IL-6 expression was statistically significant (P =

0.0124), while the differences in IFNG and IL21 transcript levels approximated significance (P =

0.0700 and P = 0.0572, respectively). Levels of IL6, IFNG, and IL21 transcripts in specimens

from GS patients did not differ significantly from those in DC or CD, except for a significant

reduction in IFNG levels relative to CD (P = 0.0222). IFNG was the only cytokine gene tested

13

that showed a significant degree of variability across the three groups (P < 0.0428; Kruskal-

Wallis test). Thus, in line with its role in Th17 cell differentiation, IL-6 is expressed at

significantly increased levels in CD mucosa, but not in GS mucosa. A concomitantly reduced

expression of IFN-γ in GS relative to CD supports the notion of a lower-level involvement of the

adaptive immune system in this condition.

Small intestinal expression of Toll-like receptors

It has been reported that the expression of Toll-like receptor 1 (TLR1), TLR2, and TLR4 is

increased in the small intestine mucosa of CD patients, lending support to the idea that innate

immune phenomena may precede and/or accompany the progression of CD and other

autoimmune conditions [22-24]. To preliminarily assess the involvement of innate immunity in

GS, we compared the expression of these molecules in fresh biopsies from the three study

groups. As illustrated in Figure 5, expression of TLR1 and TLR2, but not TLR4, mRNA was

higher in CD patients than in DC, but neither of these differences reached significance. In

contrast, relative to DC, patients with GS presented significantly higher levels of TLR2

transcripts (P = 0.0295), while transcript levels of TLR1 and TLR4 in GS were generally higher

without reaching significance (P = 0.0932 and P = 0.1274, respectively).

Mucosal expression of regulatory T-cell-associated genes

To preliminarily assess whether a difference in immune regulatory function may account for the

differential involvement of the adaptive system in CD versus GS patients, we measured mRNA

expression of two regulatory T-cell (Treg) signature molecules, forkhead box P3 (FOXP3) and

transforming growth factor-β1 (TGFB1), in small intestinal biopsy specimens. In contrast to

14

earlier reports [25, 26], the levels of expression of both FOXP3 and TGFB1 in CD patients did

not differ significantly from those in DC (P = 0.8868 and P = 0.1535, respectively). As for

TGFB1, there appeared to be a trend toward lower, rather than higher, levels of expression in CD

patients (Figure 6). This phenomenon was more accentuated in GS patients, in whom expression

of FOXP3 and TGFB1 was, significantly for FOXP3, reduced relative to DC (P = 0.0325 and P

= 0.0710, respectively). FOXP3 reduction in GS accounted for a significant variation across

groups (P < 0.0250; Kruskal-Wallis test) and was also significant relative to CD (P = 0.0293),

while no significant difference was observed in TGFB1 expression in GS versus CD patients (P

= 0.7832). These findings, quite opposite to our starting hypothesis but perhaps in line with

earlier reports in CD and other conditions [27, 28], may suggest a reduced level of activation

and/or recruitment of Treg cells in the GS small intestine mucosa relative to gluten-tolerant

controls.

Discussion

CD, an autoimmune enteropathy, results from an inappropriate T-cell-mediated adaptive immune

response against ingested gliadin. In the past few years, though, it has become apparent that

“classic” CD represents the tip of the iceberg of an overall disease burden [4, 29]. An emerging

problem is the clinical characterization of a group of gluten-reactive patients, accounting for

roughly 10% of the general population, presenting with symptoms similar to CD but with

negative CD serology and histopathology. As in CD, these patients, here and elsewhere referred

to as GS [15], experience distress when eating gluten-containing products and show

improvement when following a gluten-free diet. Differently from CD, though, in GS the adverse

reactions that develop while eating gluten are not followed by the appearance of autoantibodies

15

and by persisting damage to the small intestine. Symptoms in GS may resemble some of the

gastrointestinal symptoms that are associated with CD or wheat allergy, but objective diagnostic

tests for this condition are currently missing. Therefore, a diagnosis of GS is commonly made by

exclusion.

In itself, the absence of autoantibodies and intestinal lesions does not rule out the intrinsic

toxicity of gluten, whose intake, even in non-CD individuals, has been associated with damage to

other tissues, organs and systems besides the intestine [6, 30, 31]. In the present study, we have

sought to identify functional, morphologic and immunologic parameters to help differentiate GS

from CD and to preliminarily understand its pathophysiology. We report here for the first time

evidence of differential intestinal mucosal responses to gluten in these two conditions.

We have shown that a normal to mild histology in GS is paralleled by a conserved barrier

function. Indeed, small intestinal permeability, when tested with a LA/MA double sugar probe,

was significantly lower in GS than in CD patients or even DC. Increased intestinal permeability

is thought to be an early biological change that precedes the onset of several autoimmune

diseases [32-34]. Loss of intestinal barrier function brings with it a continuous aberrant passage

of antigens across the intestinal epithelium. This may cause a switch from tolerance to immunity,

hence representing an increased risk for autoimmune and allergic diseases in individuals whose

other genetic determinants, MHC and non-MHC, give rise to inappropriate antigen processing

and presentation. In the intestinal epithelium, paracellular permeability is regulated by

intercellular TJ proteins. As recently shown, CLDNs are integral TJ components that are critical

for maintaining cell-cell adhesion in epithelial monolayers [35-37]. The overall balance of

CLDN species expressed in a particular cell type help to define the characteristics of its TJ. For

instance, CLDN1 and CLDN4 are postulated to decrease, whereas CLDN2 is postulated to

16

increase, TJ-dependent permeability [35]. In line with this notion, and with the appreciation of

reduced small intestinal permeability in GS patients, we have shown here that the GS mucosa

expresses significantly higher levels of transcripts for CLDN4 relative to CD or DC. In contrast,

other CLDN genes and other genes associated with TJ function measured in this study did not

appear to be expressed differently in the GS or CD mucosa compared to controls. Together, these

findings suggest that the distinct clinical and serological features between GS and CD patients

are associated with marked differences in mucosal barrier function and with apparent differences

in the expression of CLDN4, which encodes for a critical TJ component [38]. Further studies are

required to compare the distribution and assembly of this and other TJ proteins in these

conditions.

Patients with GS do not present significant autoimmune or allergic comorbidities, and, as

we also have shown here, the serology for common autoantibodies, including anti-tTG IgA, is

negative. Interestingly, AGA to IgA and IgG were positive in almost 50% of cases. Similarly,

higher than expected titers of AGAs, signs and symptoms associated with non-CD gluten

sensitivity, have also been reported for schizophrenia [39] and autism spectrum disorders [40].

While in CD there is a strong genetic association with the class II MHC haplotype, with about

95% of patients carrying HLA-DQ2 and the remaining 5% carrying HLA-DQ8, we have shown

that only about 50% of patients with GS carry HLA-DQ2 and/or HLA-DQ8, a percentage

slightly higher than that in the general population. This suggests a reduced level of involvement

of MHC-dependent adaptive immune responses in GS relative to CD. We have further shown

that the GS mucosa contains increased numbers of CD3+ IELs, even though these numbers were

significantly lower than those in active CD patients in the context of relatively conserved villous

architecture, corresponding to the 0 and 1 stages of the Marsh classification. This is in line with a

17

more limited involvement of the adaptive immune system in GS and may explain why this

condition is not accompanied by significant autoimmune phenomena.

In CD, an adaptive response has been shown to be triggered by tTG-deamidated gluten

peptides bound to DQ2 or DQ8. This involves the mucosal recruitment and activation of Th1 and

Th17 clones and the production of Th1- and Th17-associated cytokines, namely, IFN-γ and IL-

17A, which contribute to disrupting barrier function and initiating tissue damage [16, 18-21, 41].

In an earlier report, we showed that IL-17A transcripts are expressed at significantly higher

levels in the small intestine mucosa of at least a subgroup of CD patients, but not in GS patients

[15]. In this study, we have extended this finding and show that the Th1 signature cytokine, IFN-

γ, also is expressed at significantly lower levels in the GS versus CD mucosa. Moreover, in CD

but not in GS, we observed a significantly enhanced expression of IL-6, a pleiotropic cytokine

that is known to promote the differentiation and function of Th17 cells, as well as a similar trend

in the expression of IL-21, consistent with its established role in the pathophysiology of Th1 and

Th17 cells.

These findings might indicate that GS is an inflammatory condition mostly supported by

innate immune mechanisms. Among these, TLRs represent a family of evolutionarily conserved

receptors able to detect microbial invasion via pattern recognition and mediate a rapid

inflammatory response which may or may not progress into an antigen-dependent adaptive

response. Different combinations of TLRs are expressed in hematopoietic cells and

nonhematopoietic cells such as intestinal epithelial cells [42-44]. In this study, we have observed

that small intestine expression of TLR2, and to a lesser extent TLR1 but not TLR4, is increased

in GS patients. In the absence of markers of adaptive immunity, as we have seen, this suggests a

prevalent role of the innate immune system in the pathogenesis of GS.

18

Taken together, these findings support the idea that the prevalent involvement of innate

versus adaptive immune pathways may help explain the clinical and serological differences in

GS versus CD patients. Reduced function of Treg cells, and specifically of “adaptive” Treg cells,

has been proposed to account for the loss of immune homeostasis and the development of

autoimmune responses in CD and related conditions [26]. It might be inferred, then, that Treg

could efficiently prevent progression to this response in GS patients. A significantly reduced

mucosal expression of the distinctive Treg marker, FOXP3, as appreciated in GS patients in this

study, is therefore surprising and counterintuitive in the light of these considerations. However,

at least as surprisingly, in several studies FOXP3 and other Treg-expressed molecules, such as

TGFB1, have been found to be upregulated in the peripheral blood and intestinal mucosa of

patients with CD and related conditions, for example, type 1 diabetes [28, 45]. Analogous

findings have been reported in other conditions associated with the extensive involvement of

adaptive immunity, such as allergy and asthma, leading to speculation that a compensatory

expansion and/or mobilization of Treg might take place concomitantly with the buildup of an

adaptive effector response [46]. Paradoxically, then, if this assumption is true, a reduced

expression of Treg markers in GS might be interpreted in the context of a generally reduced

activation of adaptive immunity relative to CD. While more studies obviously are needed to

elucidate this issue, a better understanding of Treg function in CD and related conditions will

help characterize the possible pathogenetic role of reduced Treg activation and/or recruitment in

GS.

Conclusions

19

The results of this study suggest that CD and GS are distinct clinical entities caused by different

intestinal mucosal responses to gluten. CD results from a complex, as yet undetermined,

interplay of increased intestinal permeability, mucosal damage, environmental factors in addition

to gluten, and genetic predisposition, which involves both MHC and non-MHC genes. The

typical intestinal lesions in CD are thought to be mediated by both innate and adaptive immune

effector pathways. Our findings suggest that, in a different way, GS is associated with prevalent

activation of an innate immune response. Although the mechanisms responsible for the loss of

intestinal barrier function in CD have been delineated in part, the factors responsible for the loss

of gluten tolerance and the development of autoimmunity in this condition are still incompletely

understood. We believe that this study could contribute to the clinical characterization of GS as a

condition associated with prevalent gluten-induced activation of innate immunity in the absence

of detectable changes in mucosal barrier function, and that it provides additional clues to the

definition of the complex gluten-induced changes in TJ regulation and immune processes

underlying CD pathogenesis. Double-blind, placebo-controlled studies are necessary to further

solidify the definition of GS patients and to search for specific biomarkers for a proper diagnosis.

Abbreviations

CD, celiac disease; CLDN, claudin; DC, dyspeptic controls; EMA, endomysium antibodies; GS,

gluten sensitivity; IEL, intraepithelial lymphocytes; IFN, interferon; IL, interleukin; LA,

lactulose; MA, mannitol; MHC, major histocompatibility complex; OCLN, occluding; PCR,

polymerase chain reaction; TGF, transforming growth factor; Th, T helper; TJ, tight junction;

TLR, Toll-like receptor; Treg, regulatory T cell; tTG, tissue transglutaminase.

20

Competing interests

AF is stockholder of ALBA Therapeutics (Baltimore, MD, USA). AS, KML, VC, MC, MTG,

MD, RS, GM, CT, AP, MIR, PE, FF, MC, GR and LDM have no competing interests to declare.

Authors’ contributions

AS, GM and AF conceived the study. AF supported the study. AS, KML, VC, MTG, GM, MC,

LDM and AF contributed to designing the study. AS and KL carried out most of the molecular

genetics studies. MC carried out the molecular assay of Toll-like receptor pathways. MD, CT

and GR participated in sequence alignment studies. RS and FF carried out the

immunohistochemical assays; MIR and PE participated in the recruitment of the patients and

carried out the endoscopy procedures. AS, KML, VC and AF were involved in the analysis and

interpretation of the data. AS, KML, VC, GM and AF drafted the manuscript. AS, KML, VC,

MD, CT, MC, GR, LDM and AF critically revised the manuscript. All authors gave their final

approval of the version of the manuscript to be published.

Acknowledgements

The authors thank Dr. D. Siniscalco, Dr. P. Pezzella, Dr. A. Papparella and Mr. M. Mangione for

technical help and unconditional encouragement. These studies were partially supported by

National Institutes of Health grant R21DK078699 to AF and by Giunta Regionale Campania

Assessorato alla Ricerca Scientifica L.R. 5 28/03/2002 to MC.

21

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25

Table 1. Clinical and laboratory characteristics of the study subjectsa

aDC, dyspeptic controls; CD, celiac disease; GS, gluten-sensitive individuals; SD, standard deviation; EMA-IgA,

endomysium antibodies-immunoglobulin A; tTG-IgA, tissue transglutaminase-immunoglobulin A; AGA-IgA/IgG,

anti-gliadin antibodies-immunoglobulin A/immunoglobulin G; MHC, major histocompatibility complex; IgE,

immunoglobulin E.

Characteristics DC CD GS

Number of samples 39 42 26

Age at diagnosis, yr

(mean ± SD)

33.36 ± 10.94 26.88 ± 11.77 30.73 ± 12.19

Sex (F/M) 23/16 32/10 17/9

Intestinal symptoms

Dyspepsia Chronic diarrhea

Abdominal pain

Weight fluctuation

Weakness

Smelly, fatty stools

Diarrhea

Abdominal pain

Weight loss

Gas

Extraintestinal

symptoms

None Bone or joint pain

Osteoporosis

Behavioral changes

Tingling leg numbness

Muscle cramps

Missed menstruation

Infertility

Recurrent miscarriage

Delayed growth

Thyroiditis

Tooth discoloration

Unexplained anemia

Bone or joint pain

Osteoporosis

Leg numbness

Muscle cramps

Unexplained anemia

Glossitis

EMA-IgA All negative 34 positive

3 negative

5 not determined

All negative

tTG-IgA All negative 37 positive

5 negative

All negative

AGA-IgA/IgG All negative 29 positive

10 negative

3 not determined

12 positive

13 negative

1 not determined

MHC profile None determined 22 DQ2 and/or DQ8 positive

2DQ2 and/or DQ8 negative

18 not determined

12 DQ2 and/or DQ8 positive

9 DQ2 and/or DQ8 negative

Wheat IgE 1 positive

37 negative

1 not determined

18 negative

24 not determined

5 not determined

22 negative

4 not determined

26

Figure legends

Figure 1. Reduced intestinal permeability in gluten-sensitive (GS) patients. Small intestinal

permeability was probed by measuring the urinary cumulative 5-hour amount of lactulose (LA)

(percentage of ingested), mannitol (MA) and the LA-to-MA ratio as described in the Methods.

Boxes represent the medians and interquartile ranges, and whiskers represent the range, of

independent determinations in 13 GS patients, 11 celiac disease (CD) patients, and 14 dyspeptic

controls (DC) (see intestinal permeability section of the text, pages 8-9). *P < 0.05 relative to

DC and †P < 0.05 relative to CD (Mann-Whitney U test).

Figure 2. The relative expression of tight junction (TJ)-related genes in the mucosa of gluten-

sensitive patients (GS) versus celiac disease patients (CD). Expression of the indicated genes

encoding for components of the TJ complex was measured by quantitative polymerase chain

reaction assay of RNA extracted from small intestinal bioptic specimens (see Methods). Boxes

represent the median (interquartile ratio) and whiskers represent the range of relative RNA

levels, expressed as a percentage of an 18S housekeeping gene in 24 GS patients, 31 CD patients

and 24 dyspeptic controls. *P < 0.05 relative to CD (Mann-Whitney U test).

Figure 3. Reduced numbers of intraepithelial lymphocytes (IELs) in gluten-sensitive patients

(GS) versus celiac disease patients (CD). Small intestinal bioptic specimens were stained for the

T-cell marker CD3 in immunohistochemistry, and CD3+ cells are enumerated as indicated in

Methods. Boxes represent the median (interquartile range) and whiskers represent the range of

IEL numbers relative to 100 enterocytes in the same samples in 16 GS, 11 CD and 12 dyspeptic

27

controls (DC). ***P < 0.0005 relative to DC; †††

P < 0.0005 relative to CD (Mann-Whitney U

test).

Figure 4. Increased levels of transcripts for effector cytokines in the mucosa from celiac disease

patients (CD), but not gluten-sensitive patients (GS). Expression of the indicated genes was

measured by quantitative polymerase chain reaction assay (see Methods). Boxes represent the

median (interquartile range) and whiskers represent the range of RNA levels relative to an 18S

housekeeping gene in 13 GS, 19 CD and 11 dyspeptic controls (DC). *P < 0.05 relative to DC

(Mann-Whitney U test).

Figure 5. Increased levels of transcripts for pattern recognition receptors in the mucosa from

gluten-sensitive patients (GS), but not celiac disease patients (CD). Expression of the indicated

genes encoding for Toll-like receptors TLR1, TLR2 and TLR4 was measured by quantitative

polymerase chain reaction assay (see Methods). Boxes represent the median (interquartile range)

and whiskers represent the range of RNA levels relative to an 18S housekeeping gene in 8 GS,

12 CD and 5 dyspeptic controls (DC). *P < 0.05 relative to DC (Mann-Whitney U test).

Figure 6. Reduced levels of transcripts of immune regulatory genes in the mucosa from gluten-

sensitive patients (GS), but not celiac disease patients (CD). Expression of the indicated genes

was measured by quantitative polymerase chain reaction assay (see Methods). Boxes represent

the median (interquartile range) and whiskers represent the range of RNA levels relative to an

18S housekeeping gene in 19 GS, 12 CD and 8 dyspeptic controls (DC). *P < 0.05 relative to DC

(Mann-Whitney U test).

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

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